Multiple sclerosis (MS) is the most common non-traumatic neurological disorder affecting young adults. Yet because both symptoms and conventional MR imaging findings of MS are nonspecific and do not always correspond to each other, prompt diagnosis of disease activity and treatment follow-up present significant challenges. Furthermore, current therapies for MS are only partially effective and patients often have breakthrough disease activity. Myeloperoxidase (MPO) is a highly oxidizing enzyme abundantly secreted by active macrophages/microglial cells and certain monocytes in inflammation, and is found in active inflammatory MS lesions. We have recently shown that the MR imaging agent, bis-5HT-DTPA-Gd (MPO-Gd) is highly specific and sensitive to MPO activity in in vitro and in vivo experiments. We have found that MPO-Gd to be nontoxic, possesses high Gd stability, and is well tolerated by animals even at high doses. We found that MPO-Gd imaging can detect and confirm preclinical disease, and can identify 40% more and 40% smaller lesions compared to conventional MR contrast imaging in the mouse experimental autoimmune encephalomyelitis (EAE) model. We and others have also found that myeloperoxidase activity is highly associated with areas of microglia/macrophage infiltration and demyelination both in MS and animal models of MS. Preliminary studies administering an MPO-specific inhibitor in mice induced with EAE reduced inflammatory cell recruitment and ameliorated symptoms. Therefore, we hypothesize that MPO is not only an ideal biomarker to track MS disease activity, but may also play an important role in the pathogenesis of demyelination. As current immunomodulatory therapies for MS target predominately lymphocytes, targeting macrophage/microglial inflammation by modifying the end product of macrophage/microglia function (i.e., MPO activity), may represent a new area for treating MS. In addition, this strategy may be combined with therapies targeting lymphocytes to potentially achieve synergistic beneficial effects, thus allowing lower doses of each therapy to be used to decrease drug toxicity. Therefore, using the EAE mouse model, this proposal aims to 1) establish MPO as a biomarker for inflammatory demyelination by tracking and modulating MPO activity as the disease exacerbates and remits, correlating imaging, biochemical, and histopathological changes to inflammatory and anti-inflammatory markers and immune cell populations shifts, and 2) establish MPO-Gd imaging as a superior method for tracking treatment effects, and design novel synergistic therapeutic regimen to treat both lymphocytic and macrophage/microglial inflammation. We expect the results of this proposal will validate MPO as an imaging biomarker and treatment target, change drug development strategies that are currently focused on lymphocytes and validated using nonspecific conventional imaging, and set the stage for future translation of MPO-Gd imaging and MPO treatment to human MS.